Creation of a genome-wide metabolic pathway database for Populus trichocarpa using a new approach for reconstruction and curation of metabolic pathways for plants

Abstract

Metabolic networks reconstructed from sequenced genomes or transcriptomes can help visualize and analyze large-scale experimental data, predict metabolic phenotypes, discover enzymes, engineer metabolic pathways, and study metabolic pathway evolution. We developed a general approach for reconstructing metabolic pathway complements of plant genomes. Two new reference databases were created and added to the core of the infrastructure: a comprehensive, all-plant reference pathway database, PlantCyc, and a reference enzyme sequence database, RESD, for annotating metabolic functions of protein sequences. PlantCyc (version 3.0) includes 714 metabolic pathways and 2,619 reactions from over 300 species. RESD (version 1.0) contains 14,187 literature-supported enzyme sequences from across all kingdoms. We used RESD, PlantCyc, and MetaCyc (an all-species reference metabolic pathway database), in conjunction with the pathway prediction software Pathway Tools, to reconstruct a metabolic pathway database, PoplarCyc, from the recently sequenced genome of Populus trichocarpa. PoplarCyc (version 1.0) contains 321 pathways with 1,807 assigned enzymes. Comparing PoplarCyc (version 1.0) with AraCyc (version 6.0, Arabidopsis [Arabidopsis thaliana]) showed comparable numbers of pathways distributed across all domains of metabolism in both databases, except for a higher number of AraCyc pathways in secondary metabolism and a 1.5-fold increase in carbohydrate metabolic enzymes in PoplarCyc. Here, we introduce these new resources and demonstrate the feasibility of using them to identify candidate enzymes for specific pathways and to analyze metabolite profiling data through concrete examples. These resources can be searched by text or BLAST, browsed, and downloaded from our project Web site (http://plantcyc.org).

Figure 1.

Comparison of PoplarCyc and AraCyc. A, Total number of pathways in each metabolic domain. B, The number of enzymes that catalyze the reactions in pathways within each metabolic domain in the database. Because some reactions are part of more than one domain, the sum of the percentages for all of the metabolic domains will be greater than 100%. C, Percentage of reactions without any associated enzymes in each metabolic domain. “Others” includes alcohols, aldehydes, amines and polyamines, aromatic compounds, C1 compounds, carboxylates, inorganic nutrients, and unclassified.

Figure 2.

Using PlantCyc to find a candidate enzyme in cowpea. The ureide biosynthesis pathway shows data for several experimentally verified enzymes from soybean, shown in boldface (http://www.plantcyc.org:1555/PLANT/new-image?type=PATHWAY&object=URSIN-PWY&detail-level=2&EXP-ONLY=T). Clicking on the gene encoding the last enzyme in the pathway, HIUHase (A), opens a gene detail page that has a link to the Entrez gene database at the National Center for Biotechnology Information (B). The link provides access to the sequence deposited in GenBank. The BLAST tool can be accessed directly from this page (C). A TBLASTX analysis performed against the nonhuman, nonmouse EST data set (est_others) using the default parameters and with the species search limited to cowpea (taxid: 3917) yielded the top result shown in D. A BLAST query using the putative protein encoded by this cDNA against the RESD confirmed that HIUHase was the best hit within this data set.

Figure 3.

Displaying metabolite data on the Omics Viewer. The Omics Viewer is displaying the ratio of selected metabolite levels in gai-expressing transgenic poplar leaves to wild type (WT) poplar (Populus tremula × Populus alba) leaves (for details, see Busov et al., 2006). The underlying raw data for GA levels were measured in units of ng g⁻¹ dry weight, whereas all other compound measurements were made in units of μg Glc equivalent g⁻¹ fresh weight. Pathways with increased and decreased relative levels of important compounds can easily be identified on the overview (A). For instance, the pathways associated with GA biosynthesis and inactivation each have several compounds with altered abundances (B and C). Clicking on any compound in the overview brings up an associated pathway popup window, as shown for the “gibberellin biosynthesis III” pathway (B) as well as for the two cell wall-related pathways that each has one compound with altered levels (D and E). These more detailed diagrams identify the enzymes and compounds depicted using shapes and lines on the Omics Viewer. Hyperlinks from the popup window to the relevant pathway and compound pages in PoplarCyc allow access to more detailed information, such as curated pathway summaries, enzyme sequence information, etc. A data table and a list of compounds from the input file that do not match any current entries in PoplarCyc are displayed below the metabolic overview (F). The color key to the right (G) indicates the magnitude of the difference in relative metabolite abundance (gai versus the wild type).